Montreal Heart Institute, Université de Montréal, Montreal, Quebec, Canada H3C 3J7.
Cell Signal. 2010 Oct;22(10):1502-12. doi: 10.1016/j.cellsig.2010.05.019. Epub 2010 Jun 4.
p38 MAP kinase (MAPK) isoforms alpha, beta, and gamma, are expressed in the heart. p38alpha appears pro-apoptotic whereas p38beta is pro-hypertrophic. The mechanisms mediating these divergent effects are unknown; hence elucidating the downstream signaling of p38 should further our understanding. Downstream effectors include MAPK-activated protein kinase (MK)-3, which is expressed in many tissues including skeletal muscles and heart. We cloned full-length MK3 (MK3.1, 384 aa) and a novel splice variant (MK3.2, 266 aa) from murine heart. For MK3.2, skipping of exons 8 and 9 resulted in a frame-shift in translation of the first 85 base pairs of exon 10 followed by an in-frame stop codon. Of 3 putative phosphorylation sites for p38 MAPK, only Thr-203 remained functional in MK3.2. In addition, MK3.2 lacked nuclear localization and export signals. Quantitative real-time PCR confirmed the presence of these mRNA species in heart and skeletal muscle; however, the relative abundance of MK3.2 differed. Furthermore, whereas total MK3 mRNA was increased, the relative abundance of MK3.2 mRNA decreased in MK2(-/-) mice. Immunoblotting revealed 2 bands of MK3 immunoreactivity in ventricular lysates. Ectopically expressed MK3.1 localized to the nucleus whereas MK3.2 was distributed throughout the cell; however, whereas MK3.1 translocated to the cytoplasm in response to osmotic stress, MK3.2 was degraded. The p38alpha/beta inhibitor SB203580 prevented the degradation of MK3.2. Furthermore, replacing Thr-203 with alanine prevented the loss of MK3.2 following osmotic stress, as did pretreatment with the proteosome inhibitor MG132. In vitro, GST-MK3.1 was strongly phosphorylated by p38alpha and p38beta, but a poor substrate for p38delta and p38gamma. GST-MK3.2 was poorly phosphorylated by p38alpha and p38beta and not phosphorylated by p38delta and p38gamma. Hence, differential regulation of MKs may, in part, explain diverse downstream effects mediated by p38 signaling.
p38 MAP 激酶 (MAPK) 同工型α、β 和γ在心脏中表达。p38α 似乎具有促凋亡作用，而 p38β 则具有促肥大作用。介导这些不同作用的机制尚不清楚；因此，阐明 p38 的下游信号转导将有助于我们进一步了解。下游效应物包括 MAPK 激活的蛋白激酶 (MK)-3，它在许多组织中表达，包括骨骼肌和心脏。我们从鼠心中克隆了全长 MK3（MK3.1，384 个氨基酸）和一种新的剪接变体（MK3.2，266 个氨基酸）。对于 MK3.2，外显子 8 和 9 的跳跃导致翻译外显子 10 的前 85 个碱基对发生移码，随后出现无义终止密码子。在 3 个 p38 MAPK 的潜在磷酸化位点中，只有 Thr-203 在 MK3.2 中保持功能。此外，MK3.2 缺乏核定位和输出信号。实时定量 PCR 证实了这些 mRNA 物种在心和骨骼肌中的存在；然而，MK3.2 的相对丰度不同。此外，尽管总 MK3 mRNA 增加，但 MK2(-/-) 小鼠中 MK3.2 mRNA 的相对丰度降低。免疫印迹显示心室裂解物中有 2 条 MK3 免疫反应性条带。异位表达的 MK3.1 定位于细胞核，而 MK3.2 分布于整个细胞；然而，MK3.1 在响应渗透胁迫时易位到细胞质，而 MK3.2 则降解。p38α/β 抑制剂 SB203580 可阻止 MK3.2 的降解。此外，用丙氨酸取代 Thr-203 可防止渗透胁迫后 MK3.2 的丢失，用蛋白酶体抑制剂 MG132 预处理也可防止丢失。在体外，GST-MK3.1 被 p38α 和 p38β 强烈磷酸化，但 p38δ 和 p38γ 的磷酸化能力较差。GST-MK3.2 被 p38α 和 p38β 磷酸化能力差，不被 p38δ 和 p38γ 磷酸化。因此，MK 的差异调节可能部分解释了 p38 信号转导介导的不同下游作用。
